Alpha2-macroglobulin is a novel substrate for ADAMTS-4 and ADAMTS-5 and represents an endogenous inhibitor of these enzymes

Osteoarthritis is characterized by the loss of aggrecan and collagen from the cartilage extracellular matrix. The proteinases responsible for the breakdown of cartilage aggrecan include ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). Post-translational inhibition of ADAMTS-4/-5 activity may be important for maintaining normal homeostasis of aggrecan metabolism, and thus, any disruption to this inhibition could lead to accelerated aggrecan breakdown. To date TIMP-3 (tissue inhibitor of matrix metalloproteinases-3) is the only endogenous inhibitor of ADAMTS-4/-5 that has been identified. In the present studies we identify alpha(2)-macroglobulin (alpha(2)M) as an additional endogenous inhibitor of ADAMTS-4 and ADAMTS-5. alpha(2)M inhibited the activity of both ADAMTS-4 and ADAMTS-5 in a concentration-dependent manner, demonstrating 1:1 stoichiometry with second-order rate constants on the order of 10(6) and 10(5) m(-1) s(-1), respectively. Inhibition of the aggrecanases was mediated by proteolysis of the bait region within alpha(2)M, resulting in physical entrapment of these proteinases. Both ADAMTS-4 and ADAMTS-5 cleaved alpha(2)M at Met(690)/Gly(691), representing a novel proteinase cleavage site within alpha(2)M and a novel site of cleavage for ADAMTS-4 and ADAMTS-5. Finally, the use of the anti-neoepitope antibodies to detect aggrecanase-generated alpha(2)M-fragments in synovial fluid was investigated and found to be uninformative.     

Distinguishing aggrecan loss from aggrecan proteolysis in ADAMTS-4 and ADAMTS-5 single and double deficient mice

Aggrecan loss from mouse cartilage is predominantly because of ADAMTS-5 activity; however, the relative contribution of other proteolytic and nonproteolytic processes to this loss is not clear. This is the first study to compare aggrecan loss with aggrecan processing in mice with single and double deletions of ADAMTS-4 and -5 activity (Deltacat). Cartilage explants harvested from single and double ADAMTS-4 and -5 Deltacat mice were cultured with or without interleukin (IL)-1alpha or retinoic acid and analyzed for (i) the kinetics of (35)S-labeled aggrecan loss, (ii) the pattern of (35)S-labeled aggrecan fragments released into the media and retained in the matrix, (iii) the pattern of total aggrecan fragments released into the media and retained in the matrix, and (iv) specific cleavage sites within the interglobular and chondroitin sulfate-2 domains. The loss of radiolabeled aggrecan from ADAMTS-4/-5 Deltacat cartilage was less than that from ADAMTS-4, ADAMTS-5, or wild-type cartilage under nonstimulated conditions. IL-1alpha and retinoic acid stimulated radiolabeled aggrecan loss from wild-type and ADAMTS-4 Deltacat cartilage, but there was little effect on ADAMTS-5 cartilage. Proteolysis of aggrecan contributed most to its loss in wild-type, ADAMTS-4, and ADAMTS-5 Deltacat cartilage explants. The pattern of proteolytic processing of aggrecan in these cultures was consistent with that occurring in cartilage pathologies. Retinoic acid, but not IL-1alpha, stimulated radiolabeled aggrecan loss from ADAMTS-4/-5 Deltacat cartilage explants. Even though there was a 300% increase in aggrecan loss from ADAMTS-4/-5 Deltacat cartilage stimulated with retinoic acid, the loss was not associated with aggrecanase cleavage but with the release of predominantly intact aggrecan consistent with the phenotype of the ADAMTS-4/-5 Deltacat mouse. Our results show that chondrocytes have additional mechanism for the turnover of aggrecan and that when proteolytic mechanisms are blocked by ablation of aggrecanase activity, nonproteolytic mechanisms compensate to maintain cartilage homeostasis.     

Screening of potential a disintegrin and metalloproteinase with thrombospondin motifs-4 inhibitors using a collagen model fluorescence resonance energy transfer substrate

The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Type II collagen provides cartilage with its tensile strength, whereas the water-binding capacity of aggrecan provides compressibility and elasticity. Aggrecan breakdown leads to an increase in proteolytic susceptibility of articular collagen; hence, aggrecan may also have a protective effect on type II collagen. Given their role in aggrecan degradation and differing substrate specificity profiles, the pursuit of inhibitors for both aggrecanase 1 (a disintegrin and metalloproteinase with thrombospondin motifs-4 [ADAMTS-4]) and aggrecanase 2 (ADAMTS-5) is desirable. We previously described collagen model fluorescence resonance energy transfer (FRET) substrates for aggrecan-degrading members of the ADAMTS family. These FRET substrate assays are also fully compatible with multiwell formats. In the current study, a collagen model FRET substrate was examined for inhibitor screening of ADAMTS-4. ADAMTS-4 was screened against a small compound library (n=960) with known pharmacological activity. Five compounds that inhibited ADAMTS-4>60% at a concentration of 1muM were identified. A secondary screen using reversed-phase high-performance liquid chromatography (RP-HPLC) was developed and performed for verification of the five potential inhibitors. Ultimately, piceatannol was confirmed as a novel inhibitor of ADAMTS-4, with an IC(50) value of 1muM. Because the collagen model FRET substrates have distinct conformational features that may interact with protease secondary substrate sites (exosites), nonactive site-binding inhibitors can be identified via this approach. Selective inhibitors for ADAMTS-4 would allow a more definitive evaluation of this protease in osteoarthritis and also represent a potential next generation in metalloproteinase therapeutics.     

Proteolytic activities of human ADAMTS-5: comparative studies with ADAMTS-4

Aggrecanases have been characterized as proteinases that cleave the Glu373-Ala374 bond of the aggrecan core protein, and they are multidomain metalloproteinases belonging to the ADAMTS (adamalysin with thrombospondin type 1 motifs) family. The first aggrecanases discovered were ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). They contain a zinc catalytic domain followed by non-catalytic ancillary domains, including a disintegrin domain, a thrombospondin domain, a cysteine-rich domain, and a spacer domain. In the case of ADAMTS-5, a second thrombospondin domain follows the spacer domain. We previously reported that the non-catalytic domains of ADAMTS-4 influence both its extracellular matrix interaction and proteolytic abilities. Here we report the effects of these domains of ADAMTS-5 on the extracellular matrix interaction and proteolytic activities and compare them with those of ADAMTS-4. Although the spacer domain was critical for ADAMTS-4 localization in the matrix, the cysteine-rich domain influenced ADAMTS-5 localization. Similar to previous reports of other ADAMTS family members, very little proteolytic activity was detected with the ADAMTS-5 catalytic domain alone. The sequential inclusion of each carboxyl-terminal domain enhanced its activity against aggrecan, carboxymethylated transferrin, fibromodulin, decorin, biglycan, and fibronectin. Both ADAMTS-4 and -5 had a broad optimal activity at pH 7.0-9.5. Aggrecanolytic activities were sensitive to the NaCl concentration, but activities on non-aggrecan substrates, e.g. carboxymethylated transferrin, were not affected. Although ADAMTS-4 and ADAMTS-5 had similar general proteolytic activities, the aggrecanase activity of ADAMTS-5 was at least 1,000-fold greater than that of ADAMTS-4 under physiological conditions. Our studies suggest that ADAMTS-5 is a major aggrecanase in cartilage metabolism and pathology.     

ADAMTS-5 deficiency does not block aggrecanolysis at preferred cleavage sites in the chondroitin sulfate-rich region of aggrecan

In the mouse, proteolysis in the aggrecan interglobular domain is driven by ADAMTS-5, and mice deficient in ADAMTS-5 catalytic activity are protected against aggrecan loss and cartilage damage in experimental models of arthritis. Here we show that despite ablation of ADAMTS-5 activity, aggrecanolysis can still occur at two preferred sites in the chondroitin sulfate-rich region. Retinoic acid was more effective than interleukin-1alpha (IL) in promoting cleavage at these sites in ADAMTS-5-deficient cartilage. These results suggest that cleavage at preferred sites in the chondroitin sulfate-rich region is mediated by ADAMTS-4 or an aggrecanase other than ADAMTS-5. Following retinoic acid or IL-1alpha stimulation of cartilage explants, aggrecan fragments in medium and extracts contained SELE(1279) or FREEE(1467) C-terminal sequences. Some SELE(1279) and FREEE(1467) fragments were retained in the cartilage, with intact G1 domains. Other SELE(1279) fragments were released into the medium and co-migrated with the (374)ALGS neoepitope, indicating they were aggrecanase-derived fragments. In contrast none of the FREEE(1467) fragments released into the medium co-migrated with the (374)ALGS neoepitope, suggesting that, despite their size, these fragments were not products of aggrecanase cleavage in the interglobular domain. ADAMTS-5, but not ADAMTS-1, -4, or -9, was up-regulated 8-fold by retinoic acid and 17-fold by IL-1alpha treatment. The data show that whereas ADAMTS-5 is entirely responsible for cleavage in the interglobular domain, cleavage in the chondroitin sulfate-rich region is driven either by ADAMTS-4, which compensates for loss of ADAMTS-5 in this experimental system, or possibly by another aggrecanase. The data show that there are differential aggrecanase activities with preferences for separate regions of the core protein.     

Proprotein convertase activation of aggrecanases in cartilage in situ

Proteolytic degradation of the major cartilage macromolecules, aggrecan and type II collagen, is a key pathological event in osteoarthritis (OA). ADAMTS-4 and ADAMTS-5, the primary aggrecanases capable of cartilage aggrecan cleavage, are synthesized as latent enzymes and require prodomain removal for activity. The N-termini of the mature proteases suggest that activation involves a proprotein convertase, but the specific family member responsible for aggrecanase activation in cartilage in situ has not been identified. Here we describe purification of a proprotein convertase activity from human OA cartilage. Through biochemical characterization and the use of siRNA, PACE4 was identified as a proprotein convertase responsible for activation of aggrecanases in osteoarthritic and cytokine-stimulated cartilage. Posttranslational activation of ADAMTS-4 and ADAMTS-5 was observed in the extracellular milieu of cartilage, resulting in aggrecan degradation. These findings suggest that PACE4 represents a novel target for the development of OA therapeutics.     

A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers

The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood. We show that mutant ADAMTS-5 lacking the catalytic domain, but with a full suite of ancillary domains inhibits wild type ADAMTS activity, in vitro and in vivo, in a dominant-negative manner. The data suggest that mutant ADAMTS-5 binds to wild type ADAMTS-5; thus we tested the hypothesis that ADAMTS-5 associates to form oligomers. Co-elution, competition, and in situ PLA experiments using full-length and truncated recombinant ADAMTS-5 confirmed that ADAMTS-5 molecules interact, and showed that the catalytic and disintegrin-like domains support these intermolecular interactions. Cross-linking experiments revealed that recombinant ADAMTS-5 formed large, reduction-sensitive oligomers with a nominal molecular mass of ∼400 kDa. The oligomers were unimolecular and proteolytically active. ADAMTS-5 truncates comprising the disintegrin and/or catalytic domains were able to competitively block full-length ADAMTS-5-mediated aggrecan cleavage, measured by production of the G1-EGE³⁷³ neoepitope. These results show that ADAMTS-5 oligomerization is required for full aggrecanase activity, and they provide evidence that blocking oligomerization inhibits ADAMTS-5 activity. The data identify the surface provided by the catalytic and disintegrin-like domains of ADAMTS-5 as a legitimate target for the design of aggrecanase inhibitors.     

Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family.

Aggrecan is responsible for the mechanical properties of cartilage. One of the earliest changes observed in arthritis is the depletion of cartilage aggrecan due to increased proteolytic cleavage within the interglobular domain. Two major sites of cleavage have been identified in this region at Asn(341)-Phe(342) and Glu(373)-Ala(374). While several matrix metalloproteinases have been shown to cleave at Asn(341)-Phe(342), an as yet unidentified protein termed "aggrecanase" is responsible for cleavage at Glu(373)-Ala(374) and is hypothesized to play a pivotal role in cartilage damage. We have identified and cloned a novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity, ADAMTS11 (aggrecanase-2), which has extensive homology to ADAMTS4 (aggrecanase-1) and the inflammation-associated gene ADAMTS1. ADAMTS11 possesses a number of conserved domains that have been shown to play a role in integrin binding, cell-cell interactions, and extracellular matrix binding. We have expressed recombinant human ADAMTS11 in insect cells and shown that it cleaves aggrecan at the Glu(373)-Ala(374) site, with the cleavage pattern and inhibitor profile being indistinguishable from that observed with native aggrecanase. A comparison of the structure and expression patterns of ADAMTS11, ADAMTS4, and ADAMTS1 is also described. Our findings will facilitate the study of the mechanisms of cartilage degradation and provide targets to search for effective inhibitors of cartilage depletion in arthritic disease.     

Altered proteolytic activities of ADAMTS-4 expressed by C-terminal processing

ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs) is a multidomain metalloproteinase belonging to the reprolysin family. The enzyme cleaves aggrecan core protein at several sites. Here we report that the non-catalytic ancillary domains of the enzyme play a major role in regulating aggrecanase activity, with the C-terminal spacer domain masking the general proteolytic activity. Expressing a series of domain deletion mutants in mammalian cells and examining their aggrecan-degrading and general proteolytic activities, we found that full-length ADAMTS-4 of 70 kDa was the most effective aggrecanase, but it exhibited little activity against the Glu(373)-Ala(374) bond, the site originally characterized as a signature of aggrecanase activity. Little activity was detected against reduced and carboxymethylated transferrin (Cm-Tf), a general proteinase substrate. However, it readily cleaved the Glu(1480)-Gly(1481) bond in the chondroitin sulfate-rich region of aggrecan. Of the constructed mutants, the C-terminal spacer domain deletion mutant more effectively hydrolyzed both the Glu(373)-Ala(374) and Glu(1480)-Gly(1481) bonds. It also revealed new activities against Cm-Tf, fibromodulin, and decorin. Further deletion of the cysteine-rich domain reduced the aggrecanase activity by 80% but did not alter the activity against Cm-Tf or fibromodulin. Further removal of the thrombospondin type I domain drastically reduced all tested proteolytic activities, and very limited enzymatic activity was detected with the catalytic domain. Full-length ADAMTS-4 binds to pericellular and extracellular matrix, but deletion of the spacer domain releases the enzyme. ADAMTS-4 lacking the spacer domain has promiscuous substrate specificity considerably different from that previously reported for aggrecan core protein. Finding of ADAMTS-4 in the interleukin-1alpha-treated porcine articular cartilage primarily as a 46-kDa form suggests that it exhibits a broader substrate spectrum in the tissue than originally considered.     

The development and characterization of a competitive ELISA for measuring active ADAMTS-4 in a bovine cartilage ex vivo model

ADAMTS-4 (aggrecanase1) is believed to play an important role in the degradation of aggrecan during the progression of joint diseases. ADAMTS-4 is synthesized as a latent pro-enzyme that requires the removal of the pro-domain, exposing the N-terminal neoepitope, to achieve activity. We developed a monoclonal antibody against this neoepitope of active ADAMTS-4. Furthermore, we established and characterized a competitive ELISA for measuring active ADAMTS-4 form applying the specific antibody. We used this assay to profile the presence of active ADAMTS-4 and its aggrecan degradation product (NITEGE³⁷³) in a bovine cartilage ex vivo model. We found that after stimulation with catabolic factors, the cartilage initially released high levels of aggrecanase-derived aggrecan fragments into supernatant but subsequently decreased to background levels. The level of active ADAMTS-4 released into the supernatant and retained in the cartilage matrix increased continuously throughout the 21 days of the study. The activity of ADAMTS-4 on the last day of catabolic stimulation was verified in vitro by adding deglycosylated or native aggrecan to the conditioned medium. Samples of human cartilage affected by varying degrees of osteoarthritis stained strongly for active ADAMTS-4 where surface fibrillation and clustered chondrocytes were observed. This assay could be an effective tool for studying ADAMTS-4 activity and for screening drugs regulating ADAMTS-4 activation. Moreover, it could be a potential biomarker for degenerative joint disease.     

TIMP-3 inhibition of ADAMTS-4 (Aggrecanase-1) is modulated by interactions between aggrecan and the C-terminal domain of ADAMTS-4

ADAMTS-4 (aggrecanase-1) is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In this study, we developed a sensitive fluorescence resonance energy transfer peptide assay with a K(m) in the 10 microm range and utilized this assay to demonstrate that inhibition of full-length ADAMTS-4 by full-length TIMP-3 (a physiological inhibitor of metalloproteinases) is enhanced in the presence of aggrecan. Our data indicate that this interaction is mediated largely through the binding of glycosaminoglycans (specifically chondroitin 6-sulfate) of aggrecan to binding sites in the thrombospondin type 1 motif and spacer domains of ADAMTS-4 to form a complex with an improved binding affinity for TIMP-3 over free ADAMTS-4. The results of this study therefore indicate that the cartilage environment can modulate the function of enzyme-inhibitor systems and could have relevance for therapeutic approaches to aggrecanase modulation.     

Glycosaminoglycan-binding properties and aggrecanase activities of truncated ADAMTSs: comparative analyses with ADAMTS-5, -9, -16 and -18

Aggrecanases are ADAMTS (a disintegrin and metalloproteinase with thrombospondin type I motifs) proteases capable of primary (patho)physiological cleavage at specific Glu-Xaa bonds within the core protein of the hyaluronan-binding proteoglycan aggrecan. Accumulating evidence suggests that regulation of the activity of one such aggrecanase, ADAMTS-4 (or Aggrecanase-1), involves post-translational C-terminal processing (truncation) which modulates both glycosaminoglycan (GAG)-binding affinity and enzymatic activity. In the present study, we compared the effects of C-terminal truncation on the GAG-binding properties and aggrecanase activity of ADAMTS-5 (Aggrecanase-2) relative to three other ADAMTS family members, ADAMTS-9, ADAMTS-16 and ADAMTS-18. Full-length recombinant human ADAMTS-5 (M(r) approximately 85 kDa; ADAMTS-5p85) underwent autolytic cleavage during expression by CHO/A2 cells, and co-purified with C-terminally truncated (tr) isoforms of M(r) approximately 60 kDa (ADAMTS-5p60 and M(r) approximately 45 kDa (ADAMTS-5p45). All three ADAMTS-5 isoforms bound to sulfated GAGs (heparin and chondroitin sulfate (CS)). An ADAMTS-5p45 structural mimetic, terminating at Phe628 and comprising the catalytic domain, disintegrin-like domain and thrombospondin type I repeat (TSR)-1 domain (designated trADAMTS-5F628), also bound to heparin, and exhibited potent aggrecanase activity toward cleavage sites both in the aggrecan CS-2-attachment region (at Glu1771-Ala1772) and in the interglobular domain (at Glu373-Ala374). Further truncation (deletion of the TSR-1 domain) of ADAMTS-5 significantly reduced aggrecanase activity, although appreciable GAG (heparin)-binding affinity was maintained. Other TSR-1 domain-bearing truncated ADAMTS constructs demonstrating either positive GAG-binding ability (trADAMTS-9F649) or negligible GAG-affinity (trADAMTS-16F647 and trADAMTS-18F650) displayed comparably low aggrecanase activities. Thus, the presence of TSR-1 on truncated ADAMTSs appears to be necessary, but not sufficient, for effective aggrecanase-mediated catalysis of target Glu-Xaa bonds. Similarly, GAG-binding ability, irrespective of the presence of a TSR-1 domain, does not necessarily empower truncated ADAMTSs with proficient aggrecanase activity.     

High resolution crystal structure of the catalytic domain of ADAMTS-5 (aggrecanase-2)

Aggrecanase-2 (a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5)), a member of the ADAMTS protein family, is critically involved in arthritic diseases because of its direct role in cleaving the cartilage component aggrecan. The catalytic domain of aggrecanase-2 has been refolded, purified, and crystallized, and its three-dimensional structure determined to 1.4A resolution in the presence of an inhibitor. A high resolution structure of an ADAMTS/aggrecanase protein provides an opportunity for the development of therapeutics to treat osteoarthritis.     

Aggrecanases and cartilage matrix degradation

The loss of extracellular matrix macromolecules from the cartilage results in serious impairment of joint function. Metalloproteinases called 'aggrecanases' that cleave the Glu³⁷³–Ala³⁷⁴ bond of the aggrecan core protein play a key role in the early stages of cartilage destruction in rheumatoid arthritis and in osteoarthritis. Three members of the ADAMTS family of proteinases, ADAMTS-1, ADAMTS-4 and ADAMTS-5, have been identified as aggrecanases. Matrix metalloproteinases, which are also found in arthritic joints, cleave aggrecans, but at a distinct site from the aggrecanases (i.e. Asn³⁴¹–Phe³⁴²). The present review discuss the enzymatic properties of the three known aggrecanases, the regulation of their activities, and their role in cartilage matrix breakdown during the development of arthritis in relation to the action of matrix metalloproteinases.     

Aggrecan protects cartilage collagen from proteolytic cleavage

The matrix components responsible for cartilage mechanical properties, type II collagen and aggrecan, are degraded in osteoarthritis through proteolytic cleavage by matrix metalloproteinases (MMPs) and aggrecanases, respectively. We now show that aggrecan may serve to protect cartilage collagen from degradation. Although collagen in freeze-thawed cartilage depleted of aggrecan was completely degraded following incubation with MMP-1, collagen in cartilage with intact aggrecan was not. Using interleukin-1-stimulated bovine nasal cartilage explants where aggrecan depletion occurs during the first week of culture, followed by collagen loss during the second week, we evaluated the effect of selective MMP and aggrecanase inhibitors on degradation. A selective MMP inhibitor did not block aggrecan degradation but caused complete inhibition of collagen breakdown. Similar inhibition was seen with inhibitor addition following aggrecan depletion on day 6-8, suggesting that MMPs are not causing significant collagen degradation prior to the second week of culture. Inclusion of a selective aggrecanase inhibitor blocked aggrecan degradation, and, in addition, inhibited collagen degradation. When the inhibitor was introduced following aggrecan depletion, it had no effect on collagen breakdown, ruling out a direct effect through inhibition of collagenase. These data suggest that aggrecan plays a protective role in preventing degradation of collagen fibrils, and that an aggrecanase inhibitor may impart overall cartilage protection.     

ADAMTS-12 associates with and degrades cartilage oligomeric matrix protein

Loss of articular cartilage because of extracellular matrix breakdown is the hallmark of arthritis. Degradative fragments of cartilage oligomeric matrix protein (COMP), a prominent noncollagenous matrix component in articular cartilage, have been observed in the cartilage, synovial fluid, and serum of arthritis patients. The molecular mechanism of COMP degradation and the enzyme(s) responsible for it, however, remain largely unknown. ADAMTS-12 (a disintegrin and metalloprotease with thrombospondin motifs) was shown to associate with COMP both in vitro and in vivo. ADAMTS-12 selectively binds to only the epidermal growth factor-like repeat domain of COMP of the four functional domains tested. The four C-terminal TSP-1-like repeats of ADAMTS-12 are shown to be necessary and sufficient for its interaction with COMP. Recombinant ADAMTS-12 is capable of digesting COMP in vitro. The COMP-degrading activity of ADAMTS-12 requires the presence of Zn2+ and appropriate pH (7.5-9.5), and the level of ADAMTS-12 in the cartilage and synovium of patients with both osteoarthritis and rheumatoid arthritis is significantly higher than in normal cartilage and synovium. Together, these findings indicate that ADAMTS-12 is a new COMP-interacting and -degrading enzyme and thus may play an important role in the COMP degradation in the initiation and progression of arthritis.     

Mechanisms involved in cartilage proteoglycan catabolism.

The increased catabolism of the cartilage proteoglycan aggrecan is a principal pathological process which leads to the degeneration of articular cartilage in arthritic joint diseases. The consequent loss of sulphated glycosaminoglycans, which are intrinsic components of the aggrecan molecule, compromises both the functional and structural integrity of the cartilage matrix and ultimately renders the tissue incapable of resisting the compressive loads applied during joint articulation. Over time, this process leads to irreversible cartilage erosion. In situ degradation of aggrecan is a proteolytic process involving cleavage at specific peptide bonds located within the core protein. The most well characterised enzymatic activities contributing to this process are engendered by zinc-dependent metalloproteinases. In vitro aggrecanolysis by matrix metalloproteinases (MMPs) has been widely studied; however, it is now well recognised that the principal proteinases responsible for aggrecan degradation in situ in articular cartilage are the aggrecanases, two recently identified isoforms of which are members of the 'A Disintegrin And Metalloproteinase with Thrombospondin motifs' (ADAMTS) gene family. In this review we have described: (i) the development of monoclonal antibody technologies to identify catabolic neoepitopes on aggrecan degradation products; (ii) the use of such neoepitope antibodies in studies designed to characterise and identify the enzymes responsible for cartilage aggrecan metabolism; (iii) the biochemical properties of soluble cartilage aggrecanase(s) and their differential expression in situ; and (iv) model culture systems for studying cartilage aggrecan catabolism. These studies have clearly established that 'aggrecanase(s)' is primarily responsible for the catabolism and loss of aggrecan from articular cartilage in the early stages of arthritic joint diseases that precede overt collagen catabolism and disruption of the tissue integrity. At later stages, when collagen catabolism is occurring, there is evidence for MMP-mediated degradation of the small proportion of aggrecan remaining in the tissue, but this occurs independently of continued aggrecanase activity. Furthermore, the catabolism of link proteins by MMPs is also initiated when overt collagen degradation is evident.     

Selective inhibition of ADAMTS-1, -4 and -5 by catechin gallate esters.

Three mammalian ADAMTS enzymes, ADAMTS-1, -4 and -5, are known to cleave aggrecan at certain glutamyl bonds and are considered to be largely responsible for cartilage aggrecan catabolism observed during the development of arthritis. We have previously reported that certain catechins, polyphenolic compounds found in highest concentration in green tea (Camellia sinensis), are capable of inhibiting cartilage aggrecan breakdown in an in vitro model of cartilage degradation. We have now cloned and expressed recombinant human ADAMTS-1, -4 and -5 and report here that the catechin gallate esters found in green tea potently inhibit the aggrecan-degrading activity of these enzymes, with submicromolar IC50 values. Moreover, the concentration needed for total inhibition of these members of the ADAMTS group is approximately two orders of magnitude lower than that which is needed to partially inhibit collagenase or ADAM-10 activity. Catechin gallate esters therefore provide selective inhibition of certain members of the ADAMTS group of enzymes and could constitute an important nutritional aid in the prevention of arthritis as well as being part of an effective therapy in the treatment of joint disease and other pathologies involving the action of these enzymes.